Lysing paenibacillis larvae by exposure to phage

ABSTRACT

Materials and Methods for lysing a strain of  Paenibacillus larvae  that is not  P. larvae  2605, including methods for providing to an environment of a bee hive infected with the strain of  P. larvae  a lysing phage that also lyses with  P. larvae  2605.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 61/758,983, filed on Jan. 31, 2013.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 2011-67013-30169,awarded by the United States Department of Agriculture. The governmenthas certain rights in the invention.

TECHNICAL FIELD

This document relates to materials and methods for treating andpreventing American Foulbrood disease in honeybees, and moreparticularly to materials and methods for using phage to lysePaenibacillus larvae in honeybees.

BACKGROUND

Honeybees pollinate agricultural crops and native plant species aroundthe world. Without the effort of the bees, many food supplies wouldsuffer. The use of industrially imported and transported bees is not atrivial endeavor. Some large bee pollination companies have a million ormore hives. Such operations may truck hundreds of thousands of bee hivesacross the United States, e.g., to California to pollinate the almondcrop grown each year. These same hives are then trucked back across thecountry to pollinate blueberries and other crops that bloom later thanalmonds. Some people make their living from harvesting honey from theirbee hives. Many bee hives are kept by amateur bee keepers who enjoy thehobby and inadvertently help neighbors through the work of their bees.

An aggressive loss of bee hives has begun to devastate the world's beepopulation. The loss is called Colony Collapse Disorder and its entirecause is not known. Some believe it is due to systemic pesticides usedon large monoculture agricultural crops. In addition to outright deathof the hives, Colony Collapse Disorder causes hives to be weakened andmade vulnerable to a number of infections.

A long known infection suffered by bees is caused by the bacterium,Paenibacillus larvae. While the associated disease is called AmericanFoulbrood disease (AFB), it is found worldwide. Infection with P. larvaeis a serious disease of honeybees that eventually destroys the infectedhive and further infects other hives. AFB affects the earliest stages ofthe larval development, just after the eggs are hatched. The younglarvae are digested from the inside out by the bacteria. With the lossof the brood, the colony has no chance to recover.

Various treatments have been used for AFB, including antibiotics such asOxytetracycline HCl and Tylosin tetrate. The bacteria quickly becameresistant to the antibiotic, however, and residue from the chemicals hasbeen found in honey. Thus, such treatment is not acceptable to thepublic. Additionally, the introduction of antibiotics into theenvironment can have serious secondary effects, such as causing otherbacteria to develop general resistance to antibiotics.

The primary current treatment for the presence of P. larvae is burningof the hives, the bees, and the equipment used to support the beekeepingof that hive. State departments of agriculture have inspectors who testfor the presence of P. larvae, and the treatment typically is donequickly. This is a drastic treatment, however, and the industry has beenhesitant to impose regulations on the inspection and treatment of hives,or to provide any other meaningful regulations to find and addressinfections.

SUMMARY

This document is based in part on the discovery that P. larvae can belysed by introducing phage into a bee hive, such that the phage canphysically associate with and lyse the P. larvae.

In one aspect, this document features a method of deterring a firststrain of P. larvae that is not P. larvae 2605, where the method caninclude providing to an environment of a bee hive infected with thefirst strain of P. larvae an isolated lytic phage that lyses P. larvae2605 and deters productive replication of P. larvae 2605. The lysingphage can be provided by delivering food for bees that contains thelysing phage to the bee hive. Lysing of the first strain of P. larvaecan cause a hole in the peptidoglycan of a cell wall of the first strainof P. larvae and cell membrane of the first strain of P. larvae which isexternalized after internal pressure force the cell membrane outside thehole in the cell wall, leading to rupture of the cell membrane and lossof intercellular components. Rupture of the cell membrane can lead todeath of the first strain of P. larvae.

In another aspect, this document features a method of deterring a firststrain of P. larvae that is not P. larvae 2605, where the method caninclude providing to an environment of a bee hive infected with thefirst strain of P. larvae a lysing phage that lyses P. larvae 2605 andat least two other strains of P. larvae selected from the groupconsisting of ATTC Numbers 9545, 25367, 25368, 25747, 25748, and 49843.The lysing phage can be provided by delivering food for bees thatcontains the lysing phage to the bee hive. Lysing of the first strain ofP. larvae can causes a hole in the peptidoglycan of a cell wall of thefirst strain of P. larvae and cell membrane of the first strain of P.larvae which is externalized after internal pressure force the cellmembrane outside the hole in the cell wall, leading to rupture of thecell membrane and loss of intercellular components. Rupture of the cellmembrane can lead to death of the first strain of P. larvae. The firststrain of P. larvae that is not P. larvae 2605 can deter productivereplication of P. larvae 2605 and the at least two other strains of P.larvae.

In another aspect, this document features a method for treating a P.larvae infection in a honeybee, where the method can includeadministering to the honeybee a composition comprising a lytic phagethat is capable of lysing P. larvae 2605 and deterring productivereplication of P. larvae 2605, where the P. larvae infection in thehoneybee is not an infection by P. larvae 2605. The composition cancontain one or more lytic phages that are capable of lysing P. larvae2605 and at least two other strains of P. larvae selected from the groupconsisting of ATTC Numbers 9545, 25367, 25368, 25747, 25748, and 49843.The composition can include honeybee larvae food.

In still another aspect, this document features a method for reducingthe risk of P. larvae infection in a honeybee, where the method caninclude administering to the honeybee a composition comprising a lyticphage that is capable of lysing P. larvae 2605 and deterring productivereplication of P. larvae 2605, wherein the P. larvae infection is not aninfection by P. larvae 2605. The composition can contain one or morelytic phages that are capable of lysing P. larvae 2605 and at least twoother strains of P. larvae selected from the group consisting of ATTCNumbers 9545, 25367, 25368, 25747, 25748, and 49843. The composition cancontain honeybee larvae food.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of a bacteriophage.

FIG. 2 is a graphic depicting replication of a bacteriophage during alytic cycle.

FIG. 3 is a graphic that more completely depicts the replication stageduring the bacteriophage lytic cycle.

FIG. 4 is a graphic representation of a complete lysogenic cycle for abacteriophage.

FIG. 5 is a graphic representation of a lysin system.

FIG. 6 is a table indicating the efficacy of various phage against P.larvae.

FIG. 7 is a table indicating isolated phage effects as a Host Range ofPhage on P. larvae.

FIG. 8 is a table indicating isolated phage effects as a Host Range ofPhage on P. larvae.

FIG. 9 is a series of representative images of phage lysis on abacterial lawn. Letters correspond to the descriptions in Table 4 andare specified by superscripts.

FIG. 10 is a graph plotting susceptibility of P. larvae strains to phagelysis, graphed as the proportion of each bacterial strain capable ofbeing lysed by phages. P. larvae strains are grouped by formersubspecies. Bacterial strains are listed from back to front.

FIG. 11 is a series of scanning electron microscope (SEM) images ofphages, labeled as follows: A, A; B, H3S; and C, H1P. Scale bars (in thebottom black border) are 50 nm for A and 100 nm for H3S and H1P.

FIG. 12 is a graph plotting the mean proportion of larvae surviving fromthe following treatments: negative control (dashed line), food withGmBHI added (solid line), and food with water added (dotted line). Errorbars represent the standard deviation.

FIG. 13 is a graph plotting the mean proportion of larvae survivinginfection with vegetative cells from P. larvae ATCC 49843, NRRL B-3554,and isolated 2188, as indicated. Error bars represent the standarddeviation.

FIG. 14 is a graph plotting the mean proportion of larvae survivingafter infection with P. larvae spores from the following strains: ATCC49843, NRRL B-3554, and isolated 2188, as indicated. Two infectionsusing spores from 2188 were conducted—one with daily doses of spores andone with a single dose on the first day. Error bars represent thestandard deviation.

FIG. 15 is a graph plotting the mean proportion of larvae survivingafter treatment with phage cocktail #1. Larvae were fed spores, phagecocktail, spores and then phage cocktail, or phage and then spores, asindicated. Error bars represent the standard deviation.

FIG. 16 is a graph plotting the mean proportion of larvae survivingafter treatment with phage cocktail #2. Larvae were fed spores, phagecocktail, spores and then phage, or phage and then spores, as indicated.Error bars represent standard deviation.

FIG. 17 is a graph plotting the mean survival rate for a comparison ofthe average of 11 control treatments to the phage cocktail #2 treatment,and the negative control replicate that corresponds to treatment. Errorbars represent the standard deviation. Error bars are not present on thenegative control (solid line) because it represents only one replicate.

FIG. 18 is a graph plotting the proportion of deceased larvae thattested positive for P. larvae DNA by PCR and gel electrophoresis.

FIG. 19 is a pair of images of the same frame taken 5.5 weeks apartduring experimental treatment of the hive. The image on the left showsdark comb and characteristic sunken capped cells, while the image on theright is slightly lighter and has fewer sunken capped cells.

DETAILED DESCRIPTION

P. larvae (previously classified as Bacillus larvae) is a pathogen ofthe larval honeybee (Apis mellifera L.), causing a fatal disease calledAmerican foulbrood. A bacteriophage of B. larvae was first isolated bySmirnova (1953) from decaying larvae of bees killed by Americanfoulbrood. Gochnauer (1955) isolated a phage from a lysogenic culture ofP. larvae. Gochnauer's phage differed from the phage isolated bySmirnova (1954) in their ability to pass through asbestos filters, heatstability, and plaque morphology (Gochnauer, 1970). In addition todescribing certain properties of the phage isolated from the strain nowknown as B. larvae NRRL B-3553 (Gochnauer and L′Arrivee, 1969),Gochnauer (1970) presented evidence suggesting that other phages werepresent in other strains of B. larvae. This conclusion was drawn fromsensitivity tests using culture filtrates from different B. larvaecultures and lawns of many different strains. No efforts were made toisolate the different phages. Gochnauer (1970) was unable to concentrateor purify the phage from strain B-3553, and, hence, was unable toobserve the morphology or analyze the nucleic acid component of thisphage.

A phage specific for B. larvae was isolated from a soil sample from apark in Plodiv, Bulgaria (Popova et al., 1976; Valerianov et al., 1976).This phage, named L3, lysed 10 of 15 strains of B. larvae tested. It didnot lyse B. cereus or B. anthracis. A phage, termed BLA, was isolated inCzechoslovakia from several B. larvae strains obtained from combscontaining bee larvae killed by American foulbrood (Drobnikova andLudvik, 1982). All of the phage preparations from different cultureswere considered to be identical, based on the sole criterion of theirappearance in electron micrographs.

Previous studies were conducted to purify and characterize the phageisolated by Gochnauer (1955) from B. larvae NRRL B-3553. Gochnauer andL′Arrivee (1969) reported that when a culture filtrate of strain B-3553was plated on lawns of B. larvae NRRL B-3553, both large plaques (2 to 3mm) and pinpoint plaques appeared. Subculturing of both resulted in auniform plaque size (1 to 2 mm), however, and the authors concluded thatboth plaque types were caused by the same phage. Other evidence,however, indicated that strain B-3553 contains two distinct phages.

As described herein, P. larvae in honeybees can be lysed by introducingphage into a bee hive, such that the phage can physically associate withand lyse the P. larvae. One phage described herein infected all eightenvironmental P. larvae strains tested, as well as two newly isolated P.larvae (wild) strains. This phage is from an amateur beekeeper's hivesin North Las Vegas at Gilcrease Orchard. None of the phages infectedother bacteria or higher organisms. Thus, although the word “virus” canhave a negative connotation, in this case, viruses are a potential meansto control the bacterium and, thereby, treat P. larvae infection.

Using phage therapy for treating and/or preventing P. larvae infectionsin honeybees can have several advantages. For example, phages generallyhave specific targets, and thus may have a low likelihood of affectingeukaryotic host cells and natural microbiota of the eukaryotic host. Inaddition, only small doses may be needed, and they may be readilyprovided on polysaccharide biofilms, for example. Further, phages arenaturally occurring.

A first research goal of the work described herein was to characterizenewly isolated environmental and lysogenic phages, to determine whetherone phage was isolated multiple times or if multiple different phageswere isolated. Either of these cases has advantages for treating AFB.For example, if the same phage was isolated 31 times, then it iswidespread and has wide potential for treatment. If more than one phagewas isolated, then a cocktail of the multiple types could be even moreeffective as a treatment.

A second goal of the research described herein was to determine ifphages can prevent AFB infection in early stage larvae. It is believedthat this approach to the treatment of AFB is environmentally andbiologically safe. In some embodiments, this document provides methodsmethod for deterring a first strain of P. larvae that is not P. larvae2605 by providing to an environment of a bee hive infected with thefirst strain of P. larvae an isolated lytic phage that lyses P. larvae2605 and deters productive replication of P. larvae 2605. In someembodiments, this document provides methods for deterring a first strainof P. larvae that is not P. larvae 2605 by providing to an environmentof a bee hive infected with the first strain of P. larvae a lysing phagethat lyses with P. larvae 2605 and at least two other strains of P.larvae selected from the group consisting of A TCC Numbers 9545, 25367,25368, 25747, 25748, and 49843. The lysing phage may be provided bydelivering food for bees that contains the lysing phage to the bee hive.The lysing phage may be provided by delivering food for bees thatcontains the lysing phage to the bee hive and the first strain of P.larvae that is not P. larvae 2605 deters productive replication of P.larvae 2605 and the at least two other strains of P. larvae.

One general description of the mechanism of treatment of the AFB is asfollows. A bacteriophage is a virus that destroys bacteria by lysis.Several varieties exist, and each typically attacks only onespecies/strain of bacteria. Infecting phage attach themselves to thecell wall of the bacterium and inject their genetic material (e.g., acharge of DNA) into the cytoplasm of the bacterium. The DNA/RNA carriesthe genetic code of the virus, and rapid multiplication of the virustakes place inside the bacterium. The growing viruses act as parasites,using the metabolism of the bacterial cell for growth and development.Eventually the bacterial cell bursts, releasing many more virusescapable of destroying similar bacteria.

With some bacteria, notably those of the Streptococcus family, infectionby certain phages can dramatically alter pathogenicity, convertingpreviously innocuous microbes into deadly pathogenic strains. Theso-called “flesh-eating” bacteria have incorporated into the chromosomesa bacteriophage that brings with it toxic genes. Another example is thecommon inhabitant of human nasal passages, Corynebacterium diphtheria.These are relatively harmless bacteria. Just like every other livingthing, bacteria have viruses that infect them. Bacterial viruses arecalled bacteriophage, or just “phage.” Phages have two means by which toinfect bacterial cells. One is lysogeny, in which the phage DNAincorporates into the chromosome of the bacterium and becomes dormantfor many generations. At least one environmental inducer is required tocause the phage DNA to excise from the bacterial chromosome andestablish the second type of infection, the lytic phase. In this phase,the bacterium is transformed into a phage-making factory. Hundreds ofphages are produced and the bacterial cell is lysed to release them. Thereleased phage then find another host bacterium, and the processrepeats.

Until the work described herein was conducted, the only phage to bediscovered and characterized were lysogenic phage that had been inducedto become lytic. Only one report, from the 1960s, described anenvironmental presence for phages that infect P. larvae.

The work described herein was conducted to determine if native lytic P.larvae phages might exist in nature, and if any such phages would behighly infective to strains of P. larvae. Ad discussed herein, theanswer to both of these questions is yes. Well over 130 samples weretested, some related to bees and some not. From these samples, 31 werefound to be positive for phages. The samples were from all over theUnited States, and they showed patterns of infection with eight strainsof P. larvae obtained from the American Type Culture Collection (ATCC).

FIG. 2 is a graphic of the replication of a bacteriophage during thelytic cycle. Before viral infection, the cell is involved in replicationof its own DNA and transcription and translation of its own geneticinformation to carry out biosynthesis, growth and cell division. Afterinfection, the viral DNA takes over the machinery of the host cell anduses it to produce the nucleic acids and proteins needed for productionof new virus particles. Viral DNA replaces the host cell DNA as atemplate for both replication (to produce more viral DNA) andtranscription (to produce viral mRNA). Viral mRNAs are then translated,using host cell ribosomes, tRNAs and amino acids, into viral proteinssuch as the coat or tail proteins. The process of DNA replication,synthesis of proteins, and viral assembly is a carefully coordinated andtimed event. The overall process of lytic infection is diagrammed in thefigure; discussion of the specific steps follows.

FIG. 3 is a graphic representation of a more complete stage ofreplication during the lytic cycle. Many bacteriophage that have beenstudied infect E. coli. The first step in the replication of the phagein its host cell is called adsorption. The phage particle undergoes achance collision at a chemically complementary site receptors on thebacterial surface, and then adheres to that site by means of its tailfibers.

Following adsorption, the phage injects its DNA (and rarely RNA) intothe bacterial cell. The tail sheath contracts and the core is driventhrough the wall to the membrane. This process is called penetration,and it may be both mechanical and enzymatic. Phage T4 packages a bit oflysozyme in the base of its tail from a previous infection and then usesthe lysozyme to degrade a portion of the bacterial cell wall forinsertion of the tail core. The DNA is injected into the periplasm ofthe bacterium; generally it is not known how the DNA penetrates themembrane.

Immediately after injection of the viral DNA, the process called“synthesis of early proteins” is initiated. This refers to thetranscription and translation of a section of the phage DNA to make aset of proteins that are needed to replicate the phage DNA. Among theearly proteins produced are a repair enzyme to repair the hole in thebacterial cell wall, a DNAase enzyme that degrades the host DNA intoprecursors of phage DNA, and a virus specific DNA polymerase that willcopy and replicate phage DNA. During this period, the cell'senergy-generating and protein-synthesizing abilities are maintained, butthey are subverted by the virus. The result is the synthesis of severalcopies of the phage DNA.

The next step is the synthesis of late proteins. Each of the severalreplicated copies of the phage DNA can be used for transcription andtranslation of a second set of proteins called the late proteins. Thelate proteins are mainly structural proteins that make up the capsomeresand the various components of the head and tail assembly. Lysozyme isanother late protein that will be packaged in the tail of the phage andused to escape from the host cell during the last step of thereplication process.

The replication of phage parts is followed by an assembly process. Theproteins that make up the capsomeres assemble themselves into the headsand “reel in” a copy of the phage DNA. The tail and accessory structuresassemble and incorporate a bit of lysozyme in the tail plate. Theviruses arrange their escape from the host cell during the assemblyprocess.

While the viruses are assembling, lysozyme is being produced as a lateviral protein. Some of this lysozyme is used to escape from the hostcell by lysing the cell wall peptidoglycan from the inside. Thisaccomplishes the release of the mature viruses, which spread to nearbycells, infect them, and complete additional cycles. The life cycle of aT-phage takes about 25-35 minutes to complete. Because the host cellsare ultimately killed by lysis, this type of viral infection is referredto as lytic infection.

FIG. 4 is a graphic representation of a complete lysogenic cycle.Lysogenic (or “temperate”) infection rarely results in lysis of thebacterial host cell. Lysogenic viruses (e.g., lambda, which infects E.coli) have a different strategy than lytic viruses for theirreplication. After penetration, the virus DNA integrates into a specificsection of the bacterial chromosome and is replicated every time thecell duplicates its chromosomal DNA during normal cell division. Suchphage DNA is called “prophage,” and the host bacteria are said to belysogenized. In the prophage state, all the phage genes except one arerepressed, and none of the usual early proteins or structural proteinsare produced.

The one phage gene that is expressed is an important one, because itcodes for the synthesis of a repressor molecule that prevents thesynthesis of phage enzymes and proteins required for the lytic cycle. Ifthe synthesis of the repressor molecule stops or if the repressorbecomes inactivated, another enzyme encoded by the prophage issynthesized, and the enzyme then excises the viral DNA from thebacterial chromosome. The excised DNA (the phage genome) can then behavelike a lytic virus to produce new viral particles and eventually lysethe host cell. This spontaneous derepression is a rare event, occurringabout one in 10,000 divisions of a lysogenic bacterium, but it assuresthat new phage are formed that can proceed to infect other cells.

It can be difficult to recognize lysogenic bacteria, because lysogenicand nonlysogenic cells appear identical. In a few situations, however,the prophage supplies genetic information such that the lysogenicbacteria exhibit a new characteristic (new phenotype) that is notdisplayed by the nonlysogenic cell. This phenomenon is called lysogenicconversion.

In lytic systems, a protein known as holin is responsible for forming apore in the cell membrane, such that lysin proteins can target bonds inthe peptidoglycan of the cell wall that are necessary component for thewall to remain intact. Lysin thus produces holes in the cell wallpeptidoglycan, and the cell membrane is externalized after internalpressure forces it through the hole in the cell wall. This leads torupture of the membrane and loss of intercellular components, causingcell death. External lysin therapy works only on Gram+ cells, however.Gram− cells have an outer membrane covering the peptidoglycan cell wall,so lysin is not able to form a hole without a holin to degrade the cellmembrane.

FIG. 5 shows a graphic representation of a phage lysin system. When aphage is inside a bacterial cell, it needs to produce holins in orderfor the lysins to reach the cell wall peptidoglycan. Holins are smallmembrane proteins that accumulate in the membrane until, at a specifictime that is “programmed” into the holin gene, the membrane suddenlybecomes permeabilized to the fully folded endolysin. Destruction of themurein bacterial cell wall and bursting of the cell are immediatesequelae. Holins control the length of the infective cycle for lyticphages, and thus are subject to intense evolutionary pressure to achievelysis at an optimal time. Holins are regulated protein inhibitors ofseveral different kinds Each of the different circled enzymes in FIG. 5represents a different type of lysin that is specific to a differentbond within the peptidoglycan. Cleavage of any one of these bonds candegrade the cell wall. When lysin is introduced from the externalenvironment, a holin is not required but is optional.

This document provides methods for deterring (e.g., preventing orreducing productive replication of) P. larvae, such as strains of P.larvae that are not P. larvae 2605. The methods provided herein caninclude, for example, providing to an environment of a bee hive infectedwith P. larvae an isolated lytic phage that can lyse P. larvae 2605 anddeter productive replication of P. larvae 2605. In some embodiments, thephage can be contained within a composition, and can be provideddirectly to bee larvae (e.g., in larvae food, or in another compositionthat larvae can ingest) or can be applied to the bee hive or portionsthereof. Lysing of the P. larvae can cause a hole in the peptidoglycanof the cell wall, and the cell membrane of the P. larvae can be isexternalized due to internal pressure that forces the membrane throughthe hole in the cell wall, leading to rupture of the cell membrane andloss of intercellular components. Rupture of the cell membrane can leadto death of the first strain of P. larvae.

In some embodiments, the methods provided herein can include providingto an environment of a bee hive infected with P. larvae one or morelysing phages that, individually or in combination, are capable oflysing P. larvae 2605 and at least two other strains of P. larvae (e.g.,two or more strains represented by ATTC Numbers 9545, 25367, 25368,25747, 25748, and 49843). That is, one phage may be capable of lysingmore than one strain of P. larvae, or multiple (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, or more than 10) phage isolates in combination (e.g., as acocktail) may be capable of lysing more than one strain of P. larvae.

The methods provided herein also can be used to treat a P. larvaeinfection in a honeybee, or to reduce the risk of P. larvae infection ina honeybee. In some embodiments, the methods provided herein can includeadministering to a honeybee a composition containing a lytic phage thatis capable of lysing P. larvae 2605 and deterring productive replicationof P. larvae 2605, where the honeybee is not infected by P. larvae 2605.In some embodiments, a composition can contain one or more lytic phagesthat are capable of lysing P. larvae 2605 and at least two other strainsof P. larvae (e.g., strains selected from the group consisting of ATTCNumbers 9545, 25367, 25368, 25747, 25748, and 49843). Thus, acomposition may contain one phage that is capable of lysing more thanone strain of P. larvae, or multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10) phage isolates that, in combination, are capable oflysing more than one strain of P. larvae.

Any suitable number of phage can be administered. For example, a methodcan include administering at least 10³ to at least 10¹⁰ (e.g., at least10³, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, at least10⁸, at least 10⁹, or at least 10¹⁰) or more plaque-forming units (PFU)to the environment of a bee hive.

Methods of obtaining and testing bacterial samples for the presence ofphage are known in the art. As described in the Examples below, forexample, several strains of P. larvae were obtained from ATCC, and eachwas tested for lysogeny. Each strain was allowed to grow for 24 hours ina flask, then centrifuged to pellet the bacterial cells, and filtered toremove remaining cells while allowing potential phage to pass through.The filtrate was then enriched with the universal host, P. larvae 2605,to allow possible phage to propagate. No induction method was needed;when the strains were grown, phage were successfully isolated from thespent medium. As described, five of eight strains tested had lysogenicphage present (isolated phage strains A, B, C, D, and E of Table 1).

To isolate phage from environmental samples, external phage particlesfrom previous lytic cycles were used to infect P. larvae 2605. Thesewere the first phages for Paenibacillus isolated from environmentalsamples. Samples were shaken in a buffer for 4 hours, and thencentrifuged and filtered. Filtrates were then enriched with P. larvae2605 to allow potential phage to propagate. All cultures were incubatedat 37° C., as 35-37° C. is the internal temperature of the hive.Cultures with the environmental samples were allowed to grow for 24hours, then spun in a centrifuge and filtered to remove bacterial cells.The filtrates were used for initial screenings. P. larvae 2605 andputative virus filtrates were combined in a melted soft agar overlay,then poured on a nutrient containing agar and allowed to solidify. Softagar overlays contained 0.95% agar, 1% yeast extract and same medium asunderlying plates (modified Brain Heart Infusion broth; mBHI).Additional salts (CaCl₂ and MgCl₂) were added to enable phage to attachto receptors of bacterial cells. The final concentration of agar whenviruses and bacteria were added was 0.6%. Viruses capable of lysing P.larvae were indicated by the formation of small holes (plaques) in thelawn of growth. Single plaques were picked with sterile wooden sticksand inoculated into fresh media, then enriched with P. larvae. This wasdone several times to ensure purity before amplifying the phage toincrease titers. Titers can be determined by diluting the phage lysateused in the overlay procedure and counting the plaques, then calculatingthe number of phage that are present in a specific volume (1 ml).

Any of a number of types of samples can be tested for phage that may beuseful in treating P. larvae infection. These include, withoutlimitation, garden soils, pig farm soil/manure, xeriscape garden soil,desert soils (creek bank, under sagebrush, near volcanic rock, inwash/creosote), leaves, crushed flowers (rose, cilantro), soil underbee-frequented bushes, compost, crushed bee extract, soils under hive,honey (UNLV hive honey, Oregon honey, Iowa honey), hive components (wax,propilis, royal jelly, dead larvae, pollen), Gilcrease Orchard samples,scales of diseased honeybee larvae, cultures of P. larvae, Las Vegaswash water samples, Burt's Bees products, and various lipbalms/cosmetics.

Table 1 lists ATCC numbers and internal designations for various P.larvae strains. Subspecies larvae or pulvifaciens are strains thatformerly had subspecies designations but now have been determined to bethe same with no subspecies designations. It has been noted that theprevious subspecies pulvifaciens were bright orange, and one can seethat the phage now isolated on a larvae strain (2605, also referenced asATCC 9545 or ATCC 9545/NRRL 2605) infect the same subspecies better thanthe pulvifaciens strains. The larvae strains are more infectious andlethal as well.

TABLE 1 ATCC # NRRL # Other # Our # Subspecies 9545 2605 2605 larvae25367 24026 367 pulvifaciens 25368 24027 368 pulvifaciens 25747 747larvae 25748 748 larvae 49843 3685 843 pulvifaciensATCC numbers are not provided for internal designations of 3688 (whichis pulvifaciens) and 3554 (which is larvae).

Table 2 is a representative listing samples from which phage wereobtained. Positive results were obtained 30 times from 130 samplestested. The left column of Table 2 shows the name of each phage isolate,and the right column contains a description of source. Thus, phagecapable of infecting P. larvae were isolated from the environment.

TABLE 2 Virus abbreviation Source σ Burt's Bees Honey and Grape seed OilHand Cream (Beeswax, honey) IV Burt's Bees lip balm from park (regular)β Burt's Bees Radiance Body Wash (Royal jelly) V Carmex lip balm VIEnvironmental sample VII Environmental sample I Garden soil - SummerlinII Garden soil - Summerlin HU Hive sample from Iowa YH/W Hive samplefrom Iowa - honey and wax C Internal phage from P. larvae 367 (25367) BInternal phage from P. larvae 368 (25368) A Internal phage from P.larvae 3685 D Internal phage from P. larvae 747 (25747) E Internal phagefrom P. larvae 843 (49843) VIII Norway lip balm from Finn Ware atScandinavian Festival in Astoria, OR H1P Propilis from bee hive -Gilcrease Orchards H2P Propilis from bee hive - Gilcrease Orchards H3PPropilis from bee hive - Gilcrease Orchards H5P Propilis from bee hive -Gilcrease Orchards XIII Scale from infected hive H1S Soil underneath beehive - Gilcrease Orchard H2S Soil underneath bee hive - GilcreaseOrchard H3S Soil underneath bee hive - Gilcrease Orchard H4S Soilunderneath bee hive - Gilcrease Orchard H5S Soil underneath bee hive -Gilcrease Orchard PAIIS1 fl. Soil underneath bee hive - PennsylvaniaPAIS2 fl. Soil underneath bee hive - Pennsylvania III Soil underneathbee hive - UNLV

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Sources of Bacteriophage Capable of Infecting P.larvae Materials and Methods

Growth of Bacterial Strains:

The following strains of Paenibacillus larvae were used: NRRL B-2605,NRRL B-3554, NRRL B-3650, ATCC-25748, ATCC-25747, ATCC-49843,ATCC-25367, ATCC-25368, and ATCC-3688. In addition, two naturallyoccurring cultures isolated from infected hives were used: 2188 and2231. Bacteria were grown for phage propagation under the sameconditions as described by Alvarado et al. (submitted for publication,2014) in a modification of BHI broth.

Environmental Sampling Technique:

Environmental samples were obtained using alcohol flame-sterilized metalspoons and placed into sterile Whirlpac bags. Samples also werecollected remotely by individuals in other locations using the samesampling methods. After collection, samples were stored at 4° C.

Sample Sources:

Lysogenic phages were screened from all 11 strains of P. larvae.Procedures adapted from Dingman et al. (J Gen Virol 65:1101-1105, 1984)were used to obtain lysogenic bacteriophages. No special methods wereneeded to induce prophage as suggested by Mayer et al. (Appl Microbiol18:697-698, 1969) from P. larvae strains, because sufficient numbers ofphage became lytic during the growth of their host bacteria. Cells weregrown as described by Alvarado et al. (supra). The presence of phageswas determined by plaque formation on a bacterial lawn of P. larvae 2605using a soft agar overlay method (Hurst and Reynolds, “Sampling virusesfrom soil,” In: Manual of environmental microbiology, Ed. Hurst,Crawford, and McInerney, 2nd ed., American Society for MicrobiologyPress, Washington, D.C., pp. 527-534, 2002).

Environmental phages were obtained from screening various soil samples,air samples, cosmetics containing materials derived from beehives, andmaterials directly from beehives such as royal jelly, wax, propolis, andhoney. These samples were obtained from locations in Nevada, Washington,New Mexico, Oregon, Pennsylvania, New York, and Iowa. Cosmetic samplesources, obtained from traditional retail settings, included variousbrands of lip balms with or without honeybee derived additions. Acombined total of 157 samples were screened. Methods for preparingenvironmental samples are fully described in Alvarado et al. (supra).Filtrates free from bacterial contamination were used as the startingmaterial for enrichment of lytic bacteriophages capable of lysing P.larvae.

Phage Enrichment, Screening and Isolation:

Bacteriophage enrichment was achieved using standard techniques asdescribed by Hurst and Reynolds (supra). To enrich for P.larvae-specific bacteriophages, the P. larvae host strain 2605 was used.This strain was utilized because it was phage-free after testing forlysogeny using the technique described above, and it was previously usedas a host strain in phage research (Woodrow, J Econ Entomol, 35:892-895,1942). Details of phage enrichment, screening and isolation are fullydescribed in Alvarado et al. (supra).

Amplification of Phages and Determination of Phage Titers:

Phage titers were determined using the soft agar overlay techniquedescribed above. Standard methods using two plates from a dilution withresulting plaque numbers ranging from 30-300 were selected to ensurestatistical accuracy. Plaques from the chosen dilution were thencounted, counts were averaged, and titers were calculated based on thedilution (Miller, “Methods for enumeration and characterization ofbacteriophages from environmental samples,” In: Techniques in MicrobialEcology, Ed. Burlage, Atlas, Stahl, Geesey, and Sayler, OxfordUniversity Press, pp. 218-233, 1998).

Soft Agar Overlay Spot Test:

After amplification, each lysate was tested to determine its ability toform plaques on each P. larvae strain and other bacterial speciesincluding: Paenibacillus sp. isolated from a hive, Paenibacilluspolymyxa, Paenibacillus alvei, Paenibacillus lentimorbus, Paenibacilluspopillae, Escherichia coli, Shigella flexneri, Bacillus cereus, Bacillussubtilis, Bacillus anthracis, Bacillus circulans, and Chromohalobactersp. A 1 ml aliquot of sterile broth and 1 ml of an overnight culture ofa single bacterial strain were added to a tube of melted GmBHI agar(0.95%) containing 37 g BHI (Difco), 4 g dextrose (Sigma), and 1 mM eachof CaCl₂ and MgCl₂ in 1 L ddH₂O. This mixture was then poured over aGmBHI agar (1.5%) plate to create a bacterial lawn. Plates were dividedinto quadrants, with 10 μl of a single lysate spotted onto the surfaceof each quadrant creating quadruplicate testing. The ability to lyse aP. larvae bacterial strain was measured by clearing. Each phage isolatewas tested against each bacterial strain using a scale from no evidenceof lysis to complete clearing (Table 6). All host range results wererecorded by the same individual for consistency. The phages with thebroadest host range and highest intensity of lysis were of interest as apotential treatment for hives infected with AFB. Therefore, these phageswere selected for further characterization. An exception was made for apair of phages that had the same host range pattern but were isolatedfrom very different sources. Determination of the similarity withinthese pairs was of interest because they might give some indication ofgeographic distribution.

EM Grid Preparation:

To prepare a highly concentrated phage lysate, 20 identical soft agaroverlay plates were prepared by mixing P. larvae strain 2605 withsufficient phage to result in complete lysis of bacterial cells. Plateswere prepared with GmBHI (0.4% Difco glucose was added to mBHI)containing 1.5% agarose, and overlays were made of GmBHI with 0.95%agarose. These plates were incubated overnight at 37° C.

For agarose removal and filtration, 5 ml of PBS pH 7.1 was added to thesurface of each plate and was allowed to sit for 20 minutes. The toplayer of the agarose overlay was then scraped off using a sterilepipette tip, making sure the underlying medium was not disturbed. Thescraped agarose plus PBS was collected and transferred to a funnel linedwith four layers of cheesecloth to remove the agarose particles. Theresulting liquid was then filtered through a sterile 0.2 μm filter (VWRor Fisher) using vacuum filtration to remove bacterial cells.

To concentrate phages, the filtrate was distributed into 50 mlpolysulfone centrifuge tubes (VWR) and phages were pelleted bycentrifugation for 15 hours at 4° C. and 18,000×g (Beckman J2-HS). Thesupernatant was removed and the centrifuge tubes were briefly inverted,being careful to prevent the phage pellet from completely drying. Thephage pellet was gently resuspended in 1.0 ml of phage buffer, pH 7.5with a composition of 10 mM Tris-HCl, 10 mM MgSO₄, and 68 mM NaCl (Dr.Malcom Zellars), using a cut-off 1 ml sterile, disposable pipette tip,then removed from the centrifuge tube and transferred to a 1.5 mlmicrocentrifuge tube. The starting volume of approximately 100 ml wasconcentrated to a final volume of 3 ml. This concentrated phagepreparation was used to prepare grids for TEM imaging.

Using a carbon-coated copper grid (Ted Pella), 10 μl of eachconcentrated preparation was placed onto the carbon surface and allowedto sit for 10 min prior to wicking away the liquid with Whatman 541paper wedges. The grid was rinsed (2×) for 2 min with sterile filteredddH₂O, and the liquid was wicked away. The grid was stained for 2 minwith 10 μl 2% uranyl acetate (pH 4.4), and the stain was wicked awaybefore allowing the grid to air dry. Grids were sent to the CAMCORfacilities at the University of Oregon for imaging.

Results

Composition of Isolated Phages and Proportion of Phage-ContainingSamples from Each Category:

A combined total of 157 P. larvae strains, environmental samples, andcommercial samples were screened for bacteriophages capable of lysing P.larvae 2605. Of the 157 samples, 32 were found to contain lytic viralparticles (Table 3). Table 4 displays the source and current designationof the 32 isolates. There was no apparent correlation between the sourcefrom which an isolate was obtained and the effectiveness of the phageagainst strains of P. larvae. The percentage of the total samplesscreened in each category was as follows: 31% soil underneath beehives,22% internal hive samples, 19% lysogenic phage, 16% cosmetics, and 12%other environmental sources. Because the number of samples in eachcategory was not equivalent, the actual proportion of positive sampleswithin a category was different from that of proportion of total samplestested. For example, although 19% of the total phages found werelysogenic, of the 11 bacterial strains tested, over half (54.5%) of thesamples contained phages. Likewise, only 16% of the total phages werefrom cosmetics, but out of the 22 cosmetic samples screened, 5 yieldedphage (22.7%) (Table 3).

TABLE 3 Proportion of samples found to contain P. larvae phage from eachcategory Positive Samples Samples Phage Isolate Containing CategoryScreened Samples (#) Phage (%) Lysogenic Phage 11 6 54.5 Cosmetics 22 522.7 Soil Underneath Beehives 53 10 18.8 Hive Samples 44 7 15.9 OtherEnvironmental Samples 27 4 14.8

TABLE 4 Source descriptions and designations of 32 phage isolates PhageCategory Source Designation Cosmetics Hand cream (contains beeswax and σhoney) Body wash (contains royal jelly) β Lip balm #1 IV Lip balm #2 VLip balm #3 VIII Hive Scale from infected hive XIII Samples Hive samplefrom Iowa HU Hive sample from Iowa (honey and YH/W wax) Propolis frombeehive - Gilcrease H1P Orchards, Nevada Propolis from beehive -Gilcrease H2P Orchards, Nevada Propolis from beehive - Gilcrease H3POrchards, Nevada Propolis from beehive - Gilcrease H5P Orchards, NevadaSoil Soil underneath beehive - Gilcrease H1S Underneath Orchards, NevadaBeehives Soil underneath beehive - Gilcrease H2S Orchards, Nevada Soilunderneath beehive - Gilcrease H3S Orchards, Nevada Soil underneathbeehive - Gilcrease H4S Orchards, Nevada Soil underneath beehive -Gilcrease H5S Orchards, Nevada Soil underneath beehive - PennsylvaniaPAIIS1 fl Soil underneath beehive - Pennsylvania PAIS2 fl Soilunderneath beehive - Pennsylvania PAIS2 med. cl. Soil underneathbeehive - UNLV, III Nevada Soil underneath beehive - Washington WA OtherGarden soil - Summerlin, Las Vegas, I Environmental Nevada SamplesGarden soil - Summerlin, Las Vegas, II Nevada Air sample (gravityplates) - VI Las Vegas, Nevada Air sample (gravity plates) - VII LasVegas, Nevada Lysogenic Phage from ATCC-49843 A Phage Phage fromATCC-25368 B Phage from ATCC-25367 C Phage from ATCC-25747 D Phage fromATCC-49843 E Phage from wild strain 2231 F

Plaque Morphology:

Individual phage filtrates produced plaques in soft agar overlays, whichwere characterized based on size and morphology (Table 5). Plaque sizesranged and were described using set plaque diameters in the followingclassifications: pinpoint (<0.1 mm), small (0.1 mm-0.5 mm), medium (0.5mm-1.0 mm), and large (>1.0 mm). Along with size, plaques wereclassified as either turbid or clear. In one case, a turbid halosurrounded a clear plaque, and this feature was also considered forcharacterization. Plaque morphologies of the phages were as follows: 4large, clear; 4 medium, clear; 3 small-medium, clear; 1 small, clear; 1pinpoint, clear; 1 small, turbid; and 5 pinpoint, turbid. Although therewas a distribution of sizes, there were more large, clear plaques thansmall, clear plaques, and more small, turbid plaques than large, turbidplaques.

TABLE 5 Plaque morphology classification of each phage observed in softagar overlays Phage Plaque Morphology Designation Size Clarity XIIILarge Clear H1P Pinpoint Turbid WA Medium Clear HIS Pinpoint Clear FLarge Clear V* Large Clear H2S Small-medium Clear H3S Medium Clear EPinpoint Turbid H5S Medium Clear VII Pinpoint Turbid D Large ClearPA1S2 - fl. Pinpoint Turbid B Pinpoint Turbid VIII Small Turbid PAIS2 -med. cl. Medium Clear Sigma Small Clear IV Small-medium Clear VISmall-medium Clear *formed plaques with a turbid halo around a clearplaque center

Host Range Distribution:

The host range results were interpreted on a scale from no clearing tocomplete clearing. Table 6 describes the classifications and FIG. 9displays representative pictures for comparison. Phages are designatedby letters and numbers, corresponding to the source from which they wereisolated. The host range of each of the 32 isolated phages on each of 27different bacterial strains is presented in Table 7. The bacterialspecies are represented across the top and are ranked from left to rightin order of susceptibility to lysis by the 32 phages. The isolatedphages are listed on the left side of the table and are ranked from topto bottom in order of the percentage of P. larvae strains they arecapable of lysing.

TABLE 6 Spot test descriptions observed in the host range experiment.

Superscript letters (a-e) correspond to the images presented in FIG. 9.

No bacteria from genera other than Paenibacillus showed susceptibilityto the isolated phages (Table 7). Even among the Paenibacillus speciestested, only one species other than P. larvae showed any susceptibility,and it was very slight. Although this Paenibacillus species was isolatedfrom a hive infected with AFB, PCR amplification of its DNA with P.larvae specific-primers revealed that this strain is not P. larvae(Piccini et al., World J Microbiol Biotechnol 18:761-765, 2002). With anNCBI BLAST search of the PCR products, the organism did not match anyother known species of Paenibacillus. Only six of the phages were ableto very mildly infect this Paenibacillus sp.

Three phages, H1P, WA, and H1S, lysed all P. larvae strains tested, andF lysed all strains with the exception of its host strain, 2231. Inaddition, these phages with broad host ranges on P. larvae were alsohighly lytic on multiple strains (+++). One exception was XIII, whichwas highly lytic only on four P. larvae strains. The isolated lysogenicphages were generally not capable of lysing the host strain from whichthey were isolated, with the exception of D and A, and these onlyproduced +/− results.

Comparing the Susceptibility of Bacteriophage Lysis on Former P. LarvaeSubspecies larvae and P. larvae Subspecies pulvifaciens:

As visualized in FIG. 10, there was a distinct difference between thesusceptibility of strains formerly designated as P. larvae larvae or P.larvae pulvifaciens when tested with the 32 newly isolated phages.Sample variances of former P. larvae pulvifaciens and former P. larvaelarvae were 0.0237 and 0.0046, respectively. Welch's t-test determinedthe values as t=4.169 and degrees of freedom ˜5.727. Using these valuesand a t-distribution table, p=0.0087. Assuming that a statisticalsignificance is inferred when p≦0.01, there is a significant differencebetween the means of the proportion of susceptibility that each group offormer P. larvae subspecies has to the P. larvae bacteriophages. Becausethe two strains that were isolated from an infected hive were notclassified under the same former subspecies as the repository strains,they were not included in this calculation.

TABLE 7 Host range of 32 isolated P. larvae bacteriophages determined bysoft agar overlay spot tests. Results are interpreted on a scale from nolysis (blank cell) to complete lysis (black cell) as described in Table6 and visualized in FIG. 9.

Comparison of Phage Morphology using TEM:

Results for 16 phages that were confidently imaged are given based onmorphological descriptions only, and the following are the possiblefamilies of these isolated phages: 13 Siphoviridae, 1 Podoviridae, 1potential Inoviridae, and 1 potential Tectoviridae (Table 8). Even amongphages potentially classified under the same family, there are sizevariations of heads and tails. Sample images are presented in FIG. 11.

TABLE 8 Morphologies of chosen phages determined from TEM images EMImaging Comparison Phage ~Head ~Head ~Tail Desig- Head Length WidthLength Possible nation Shape (nm) (nm) (nm) Family H1P Elongated 109 55227 Siphoviridae icosahedral A Elongated 114 71 212 Siphoviridaeicosahedral WA Elongated 80 35 125 Siphoviridae icosahedral H2SSpherical 50 50 200 Siphoviridae icosahedral F Elongated 115 65 120Siphoviridae icosahedral H3S Elongated 120 61 138 Siphoviridaeicosahedral PA1S2 - Elongated 87 41 190 Siphoviridae fl. icosahedral DElongated 94 47 106 Siphoviridae icosahedral PAIS2 - Elongated 148 74185 Siphoviridae med. cl. icosahedral V Spherical 56 61 157 Siphoviridaeicosahedral VIII Spherical ND ND ND Siphoviridae icosahedral H5SSpherical 150 150 225 Siphoviridae icosahedral Sigma Spherical 128 109309 Siphoviridae icosahedral HIS Spherical 70 84  40 Podoviridaeicosahedral E No Range Inoviridae? evident from heads 200-500 IIISpherical 110 110 No Tectoviridae? icosahedral evident tailsImages were provided by the CAMCOR facilities at the University ofOregon. Measurements are based on the averages of 2-4 images. Questionmark indicates uncertainty of classification based on rarity of thefamily. Family classifications are based on descriptions of morphologyonly.

As described above, a total of 32 phages were isolated from 157 sources,suggesting that about 20% of the sources screened could yield phagescapable of lysing P. larvae. In the host range results, the lack ofclearing on other genera and only one incidence of slight clearing on aPaenibacillus sp., indicates high host specificity. As a potentialtreatment for AFB, such severe host specificity is encouraging becausethe microbial ecology of the hive is not well understood, and a lack ofspecificity could harm microbes not intentionally targeted with P.larvae phages. A spot test can be undertaken in future work tospecifically test phages on the natural honeybee microbiota.

Using the most effective phages with the broadest host range on the 11P. larvae strains, it may be possible to generate a cocktail that iscapable of lysing 100% of the strains, using as few as the top threeisolated phages (H1P, WA, and H1S). A more robust cocktail could bedesigned by testing the lysing capabilities of these isolated phages onadditional strains of P. larvae. The use of a cocktail of multiplephages, rather than a single phage, may reduce the potential fordevelopment of phage resistance. Therefore, determining selectioncriteria for the most suitable phages is important. If an arbitraryproportion of strains lysed is chosen, for example 8 out of the 11, aphage cocktail capable of lysing all 11 strains with multiple phagescapable of infecting each of the strains could be designed using 14phages. Determining the effectiveness of a cocktail consisting of these14 isolated phages will be the subject of future work in developingphage therapy as a potential treatment for AFB.

Example 2 Phage Therapy for Treating AFB in Honeybees Materials andMethods

Bacterial Strains and Phage Isolates:

The following strains of P. larvae were used: NRRL B-2605, NRRL B-3554,NRRL B-3650, ATCC-25748, ATCC-25747, ATCC-49843, ATCC-25367, ATCC-25368,and ATCC-3688. In addition, two naturally occurring cultures isolatedfrom infected hives were used: 2188 and 2231. Bacterial cultures weregrown with the same media and under the same conditions described in thephage isolation methods from Alvarado et al. (supra). The phages hadbeen previously isolated as described in Alvardo et al. (supra) and wereselected from a pool of 32 total isolates based on the broadest hostrange of P. larvae strains.

Amplification and Quantification of Phage Titers:

Phage isolates were amplified prior to use in the experimentaltreatments. The procedures for amplification and quantification of phagetiters were the same as those described by Alvarado et al. (supra).

Bacterial Cell and Spore Harvesting:

Eleven strains of Paenibacillus larvae were grown in 20 ml of GmBHI at37° C. with shaking at 100 rpm. After overnight incubation, the turbidculture was pelleted by centrifugation, the supernatant discarded, andthe cells resuspended in 200 μl sterile GmBHI broth. The concentratedcells were plated in serial dilutions using GmBHI agar plates and GmBHIsterile broth dilution blanks, and then colonies were counted todetermine the colony forming units (CFU) of the concentrate. A volume of200 μl of the concentrate was added to 1 ml of prepared larvae food,resulting in a titer of 10⁵ cells per total volume. Food was mixed byvortexing, then fed to larvae on a daily basis. New food was preparedwith freshly grown bacterial cultures daily. Approximate numbers of CFUsbeing fed to each larva were calculated according to the final titers inthe larvae food and amount of food fed to each larva per day (Table 9).Spores were prepared by first inducing sporulation then harvestingspores as described by the spore methods in Alvarado et al. (supra) withthe exception of replacing the Histopenz (Sigma) density gradient withd-Sorbitol at the same concentrations. Spore concentration wascalculated by serial dilution and plating of the final product.Calculations of spore load fed to each larva per day are given in Table9.

Phage Cocktail Preparation:

Titers per ml of the amplified single phage lysates were determined aspreviously described and were as follows: H1P, 5×10⁴; WA, 3×10⁶; F,5×10⁶; V, 4×10⁵; H2S, 10⁴; H3S, 4×10⁵; XIII, 4×10⁶; E, 10⁴; H5S, 9×10³;VII, 2×10⁶; D, 10⁶; PAIS2 fl, 9×10²; and B, 5×10⁶. Two separatecocktails were made. The first (phage cocktail #1 or PC1) contained 7phages: H1P, WA, F, V, H2S, H3S, and XIII, and the second (phagecocktail #2 or PC2) contained all 13 phages. In both cases, however, thefinal titer of combined phages was about the same (phage cocktail #1,1.8×10⁶; phage cocktail #2, 1.6×10⁶). Phage cocktail makeup wasdetermined based on host range capabilities, and represents the broadestrange of lysing capability on 11 different strains of P. larvae. Avolume of 1 ml of each lysate was combined for the final phage cocktail.The final phage concentration was both calculated from initial titersand confirmed by soft agar overlay platings done in serial dilutionafter combination. A volume of 200 μL of each cocktail was added to 1 mlof prepared larvae food prior to feeding to larvae. Calculated PFUs fedto each larva per day are listed in Table 9.

TABLE 9 Volume of food and titers of phage, bacteria, and spores fed tolarvae daily Days after Grafting Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day6 Day 7 Day 8 Volume of  10  10  20  30  40  50  50  60 0 Larvae Food(μl) Calculated # 800 800 1600 2400 3200 4000 4000 4800 0 of CFUs (anystrain) Calculated # 100 100  200  300  400  500  500  600 0 ATCC 49843Spores Calculated #  90  90  180  270  360  450  450  540 0 NRRL B- 3554Spores Calculated #  90  0  180  270  360  450  450  540 0 Isolated 2188Spores Calculated  3.00³  3.00³   6.00³   9.00³   1.20⁴   1.50⁴   1.50⁴  1.80⁴ 0 Number of PFUs in PC1 Calculated  2.67³  2.67³   5.33³   8.00³  1.07⁴   1.33⁴   1.33⁴   1.60⁴ 0 Number of PFUs in PC2

Larvae Food Preparation and Treatment:

Larvae food consisted of 14.4 ml sterile, distilled water, 4.2 g royaljelly powder (Glory Bee), 0.6 g glucose (Difco), 0.6 g fructose (Difco),and 0.2 g yeast extract (Difco) as described by Peng et al. (1992). Thesugars and yeast extract were added to the water, this mixture wasfiltered, and then UV treated for 1 h. The royal jelly powder (4.2 g)was aseptically added to the water mixture but was otherwise untreated.The mixture was made homogenous by vortexing to ensure completedispersion of the royal jelly. Food was prepared and stored at −20° C.until needed. Larvae were fed increasing amounts of food each day(Crailsheim et al., In: The Coloss Beebook, Volume 1; Standard Methodsfor Apis mellifera Research, J Apicultural Research 52:12012, 2012), asindicated in Table 9. As a negative control, larvae were fed larvae foodwithout amendments while all other larvae were fed a mixture of foodwith treatment additives. In each case, 200 μL of concentrated spores,cells, or phage cocktails were added to 1 ml of larvae food as describedabove. Larvae were given the following treatments: negative control=foodwith no additives, broth control=food with GmBHI broth added to the samedilution as other additives, water control=food amended with 200 ulsterile water, food containing NRRL B-3554 vegetative cells, foodamended with ATCC 49843 vegetative cells, food amended with isolated2188 vegetative cells, food amended with NRRL B-3554 spores, foodamended with ATCC 49843 spores, food amended with 2188 spores,prophylactic phage therapy treatments and post-infection phage therapytreatments (food amended with 200 ul phage cocktail #1 or #2). Alllarvae in the experimental phage cocktail treatments were infected withspores from P. larvae 2188. Two phage cocktails, phage cocktail #1 andphage cocktail #2, were tested in both the prophylactic andpost-infection treatment experiments.

Larvae Rearing:

Larvae were reared by methods similar to those described by Crailsheimet al. (supra). Queens were caged using plastic or metal wire mesh aboutone week prior to the intended date of grafting larvae. While the queenswere confined, the age and location of larvae in the frame were ensured.Eggs, turned to a horizontal position shortly before hatching, were thenclosely observed and the hatched larvae were grafted from the frameswithin a day after hatching. Each treatment included a correspondingnegative control consisting of larvae taken from the same frame on thesame day. Preliminary experiments were conducted by placing the graftedlarvae into 96-well plates, but later were conducted by placing graftedlarvae into sterile petri dishes (VWR) because survival rates werehigher in larvae reared with more space. It appeared that highersurvival rates were observed because the larvae food was not confined,leading to a lower chance of larval drowning. Incubation microcosms werecreated by placing 1 L of 10% glycerol in the bottom of plasticcontainers, followed by a layer of plastic support on which sat the wellplates or petri dishes. The boxes were closed with loosely fittingplastic lids, allowing the humidity to be maintained at 90% within themicrocosms. Metal trays filled with water were placed on the bottom ofthe incubator to maintain humidity within the incubator's interior at80%. The temperature was kept at 34° C. Larvae were fed daily with theamount of food indicated in Table 9. On the eighth day after grafting,larvae were removed from the petri dishes and placed on sterile filterpaper in new petri dishes outside the microcosms for pupation.

For the larvae controls, larvae were fed either unamended food, fooddiluted with GmBHI, or food diluted with water (FIG. 12). The negativecontrol data represent three replicates with n=20, 21, and 15, the GmBHIdata represent two replicates with n=20 each, and the water datarepresent two replicates with n=22 and 21.

Each experimental treatment also had a corresponding negative controlprepared on the same day from the same frame and fed unamended food.Negative control data for FIGS. 13-17 represent the average of 10control replicates with n=12 or 13. During the vegetative cell infectiontreatments, two replicates for each strain with samples sizes from 32 to49 (mean size of 45 larvae) were prepared.

Larvae were fed ATCC 49843 and NRRL B-3554 spores daily. Two differenttreatments with 2188 spores were conducted—one in which larvae were fedspores daily and one in which only one dose of spores was administeredon the first day. Spore treatment sample sizes ranged from 48-53 with amean size of 50, and all spore infection treatments were conducted induplicate.

Phage preparations were administered to larvae by adding the phagecocktails suspended in GmBHI to the larvae food (as previouslydescribed). Phage cocktail experiments were conducted in duplicate, andall phage cocktail treatment sample sizes ranged from 48-55, with a meanvalue of 51.

Daily Observations:

Larvae were viewed under a dissecting microscope (Nikon) daily andobserved for signs of life that included opening and closing spiraclesor food consumption. In the event that no movement was seen for thefirst 2 days, larvae were kept until day 3 in the event that they werealive but not producing easily visualized movement. On the third day, ifno growth or movement was observed, larvae were assumed dead andremoved. Samples of dead larvae were kept at −20° C. in 20% glycerolstocks for PCR analysis in order to determine whether bacterial DNA waspresent. The number of surviving larvae was recorded daily.

Lyophilization of Phage Cocktails:

Between 10 and 15 ml of individual amplified phage lysates werelyophilized separately (LabConco Lyophilizer). Samples were allowed tocompletely dry overnight. Once all liquid was removed, samples wereweighed and equal amounts (0.02 g) of each powdered phage preparationwere combined. This powdered mixture was easily transported to the fieldsite. Experiments to ensure phage viability after lyophilization wereconducted with reconstituted lyophilized phage. Powdered phage mixtureswere resuspended in either water or sugar syrup (8.75 g sucrose/10 mlwater) and plated to determine phage viability in diluents proposed forfield study.

Field Resuspension of Phage and Spray Treatment on Hives:

Lyophilized phage preparations were taken to the field site nearBellingham, Wash., reconstituted with 10 ml of water, and then pouredinto 400 ml of sugar syrup. After shaking to homogenize the mixture, theentire volume was sprayed directly on alternating frames in the infectedbeehive. The following day, the sugar syrup mixture had been cleaned bythe nurse bees and was no longer visible. Treatments occurred on June26, June 28, July 10, July 23, and August 6. The first two treatmentswere administered in the presence of the beekeeper and the remainingthree were conducted by the beekeeper. On each date, either odd or evennumbered frames were sprayed with the sugar syrup/phage preparation.

Hive Observations:

Frames were selected on the first treatment day for qualitativevisualization of the extent of the infection and were photographed onthe first day as well as at each subsequent treatment. Gross comparisonsof the frames were made over time, but detailed results were difficultto determine based on visualizations only. Additionally, the beekeeperreported the general state of the treated hive on a regular basis untilthe end of the treatments.

Post-Treatment Actions and Related Observations:

One month after the last phage treatment was administered, the beekeeperremoved the worst of the diseased frames and replaced them with fresh,uninfected, and unpopulated frames. By October 11, the beekeeperreported no evidence of AFB in the hive, and as of the followingJanuary, no recurrence had been reported.

Statistical Analysis:

Student T tests were performed on all treatments and controls todetermine the statistical significance of their comparisons. Asignificance value of α>0.05 was used throughout the study.

Results

Lab Experiments:

Results obtained from the control experiments are shown in FIG. 12.There was a significant difference between the survival of the negativecontrol and the water control (p=0.002), and also between the survivalof the GmBHI broth control and the water control (p=0.034), but notbetween the negative control and the GmBHI broth control (p=0.347).

Results from the vegetative cell infection treatments are shown in FIG.13. There was a significant difference in the larvae survival by day 8between the negative control larvae and those infected with P. larvaeATCC 49843 (p=0.000548), as well as between the negative control larvaeand those infected with P. larvae 2188 (p=0.00560), but not with larvaeinfected with NRRL B-3554 vegetative cells (p=0.139). The larvaeinfected with NRRL B-3554 that survived until pupation were incubateduntil pupation was complete, and the body mass was recorded for eachfully pupated bee. Compared to the control bees, the mass of theinfected bees was significantly lower (p=0.0035).

Spore infection experiments indicated a significant decrease in survivalof larvae infected with spores from any of the three bacterial strainscompared to the control (FIG. 14). There was a significant differencebetween the survival rates of the larvae infected with any of the sporesand the negative control larvae ATTC 49843 (p=1.99E-8), NRRL B-3554(p=1.79E-8), and the one dose spore infection with 2188 (p=4.97E-7), butthere was not a significant difference in the survival rates of larvaefed only one dose of 2188 spores when compared to larvae fed daily dosesof 2188 spores (p=0.102).

T-test comparisons between the larvae fed spores (FIG. 14) or vegetativecells (FIG. 13) of the same strains yielded the following: ATCC 49843vegetative cells compared to spore infection, p=0.010; NRRL B-3554vegetative cells compared to spore infection, p=0.002; 2188 vegetativecells compared to the 1-dose spore infection, p=0.384. There was asignificant difference between the survival rates of larvae infectedwith spores of either ATCC 49843 or NRRL B-3554 compared to larvaeinfected with vegetative cells of the same strains. There was not,however, a significant difference between the survival rates of larvaeby day 8 between those infected with spores or vegetative cells of 2188.

Results from phage cocktail #1 experiments are shown in FIG. 15. Therewas no statistically significant difference between the negative controland phage cocktail #1 control (p=0.077) or between the negative controland larvae infected with 1 dose of 2188 spores (p=0.045). Further, therewas no statistically significant difference between larvae infected with1 dose of 2188 spores and larvae treated with phage cocktail #1 afterinfection (p=0.031), between larvae infected with 1 dose of 2188 sporesand larvae given phage as a prophylaxis prior to infection (p=0.010),between phage cocktail #1 control and larvae treated with phage afterinfection (p=0.126), between phage cocktail #1 control and larvae givenphage as a prophylaxis prior to infection (p=0.128), or between theprophylaxis and the treatment regimens using phage cocktail #1(p=0.293). There was a significant difference between the survival oflarvae given phage cocktail versus infected with spores of 2188. Therewas not a significant difference between the survival of larvae givenphage cocktail versus the negative control. There also was a significantdifference in survival rates between both forms of phage treatment(either administered prior to or after infection) and infected larvaewithout treatment, but not between the survival of the treatmentsthemselves.

Results from the phage cocktail #2 experiments are shown in FIG. 16.T-test comparisons yield the following: comparison between the negativecontrol and phage cocktail #2, p=0.069; comparison between the phagecocktail #2 and larvae infected with spores from 2188, p=0.002;comparison between larvae infected with spores and larvae treated withphage cocktail #2 after infection, p=0.271; comparison between larvaeinfected with spores and larvae given phage cocktail #2 as aprophylactic treatment prior to infection, p=0.024; and comparisonbetween the prophylaxis and the treatment regimens using phage cocktail#2, p=0.044. Assuming α<0.05, there was a significant difference betweenthe phage cocktail #2 larvae and the infection control, but not betweenthe phage cocktail #1 larvae and the negative control. There was not asignificant difference between the infection control and the treatmentregimen, but there was a significant difference between the infectioncontrol and the prophylaxis regimens. The survival of larvae treatedwith the phage cocktail prior to infection increased by 70%, and wascomparable with the survival rates of the phage cocktail controls.

The efficacy of the two different phage cocktails was determined bycomparing the data represented in FIG. 15 and FIG. 16. T-testcomparisons yield the following: comparison between the prophylaxistreatment of phage cocktail #1 and phage cocktail #2, p=0.162; andcomparison between the treatment regimen of phage cocktail #1 and phagecocktail #2, p=0.041. Assuming α<0.05, there was a significantdifference between the different phage cocktails when used as atreatment, but not when used as a prophylaxis.

Further analysis of the anomalous, significantly lower survival with thephage cocktail #2 treatment is displayed in FIG. 17. Re-evaluation ofthe raw data revealed the corresponding negative control of the phagecocktail #2 treatment that was removed from the same frame on the sameday to be much lower than the survival of the compiled average of allnegative controls.

FIG. 18 shows the proportion of deceased larvae that tested positive forP. larvae DNA by PCR and gel electrophoresis (Piccini et al., World JMicrobiol Biotechnol 18:761-765, 2002). Larvae obtained from negativecontrol and phage cocktail control experiments (both of which had nobacteria added) showed no evidence of P. larvae DNA. About 40% of thelarvae taken from vegetative cell experiments were positive for DNA,while about 25% of the larvae taken from spore experiments were positivefor DNA. The average proportion of larvae positive for P. larvae DNAfrom phage cocktail treatments, regardless of whether phage wasadministered prior to or after spore infection, was slightly lower, at20%

Field Experiment:

Experiments to determine phage viability after lyophilization wereconducted to determine whether powered phage lysates were a practicaloption to use in a field setting. Prior to lyophilization, the averagetiter of multiple phage lysates was approximately 10⁸/ml. Afterlyophilization, the cocktails were resuspended in either sugar syrup orsterile water and the average of the resuspended cocktails wasapproximately 10⁵/ml. The resuspended phage cocktails were maintained at4° C. for one month, and titers were then determined to be approximately10⁴/ml.

Pictures were taken of the same frames each time a treatment occurred,and observations were determined by the beekeeper. Pictures revealed aslight visual improvement during the treatment process, but not acomplete eradication of the disease (FIG. 19). The comb was both darkerand has more sunken capped cells (both characteristics of AFB) in theimage taken on June 28. The beekeeper reported removing the diseasedframes and replacing them with virgin, unpopulated frames aftertreatments had ended. Four months after the initial treatment, thebeekeeper reported no visible sign of infection.

Samples were obtained after the treatment regimen ceased, and theprocedures to isolate phage as previously described (Example 1) wereconducted. It was determined that the phage from the administered phagecocktails were present in the hive after the five treatments had ended.

Taken together, these results indicate an overall improvement insurvival when phage cocktails are administered to infected honeybeelarvae. Prophylactic treatment with phage cocktail #1 was slightly moreeffective than the post infection treatment, although not significantlyso, while prophylactic treatment with phage cocktail #2 wassignificantly more effective at increasing larval survival thanpost-infection treatment. This suggests that a prophylactic regimen maybe more effective at preventing the disease than a post-infectiontreatment once a hive was already infected. Further, the higher survivalof larvae that underwent prophylactic treatment with phage cocktail #2than with phage cocktail #1 indicates that a cocktail with a greaternumber of different phages is more effective than a cocktail with fewerdifferent phages. Although only one hive was experimentally treated inthe field, the fact that the hive had no recurrence of AFB after aboutsix months is promising. Thus, the results from these preliminaryexperiments indicate that phage therapy is useful for treating AmericanFoulbrood disease.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of deterring a first strain ofPaenibacillus larvae that is not P. larvae 2605, comprising providing toan environment of a bee hive infected with the first strain of P. larvaean isolated lytic phage that lyses P. larvae 2605 and deters productivereplication of P. larvae
 2605. 2. The method of claim 1, wherein thelysing phage is provided by delivering food for bees that contains thelysing phage to the bee hive.
 3. The method of claim 2, wherein lysingof the first strain of P. larvae causes a hole in the peptidoglycan of acell wall of the first strain of P. larvae and cell membrane of thefirst strain of P. larvae which is externalized after internal pressureforce the cell membrane outside the hole in the cell wall, leading torupture of the cell membrane and loss of intercellular components. 4.The method of claim 3, wherein rupture of the cell membrane leads todeath of the first strain of P. larvae.
 5. The method of claim 1,wherein lysing of the first strain of P. larvae causes a hole in thepeptidoglycan of a cell wall of the first strain of P. larvae and cellmembrane of the first strain of P. larvae which is externalized afterinternal pressure force the cell membrane outside the hole in the cellwall, leading to rupture of the cell membrane and loss of intercellularcomponents.
 6. The method of claim 5, wherein rupture of the cellmembrane leads to death of the first strain of P. larvae.
 7. A method ofdeterring a first strain of P. larvae that is not P. larvae 2605,comprising providing to an environment of a bee hive infected with thefirst strain of P. larvae a lysing phage that lyses P. larvae 2605 andat least two other strains of P. larvae selected from the groupconsisting of ATTC Numbers 9545, 25367, 25368, 25747, 25748, and 49843.8. The method of claim 7, wherein the lysing phage is provided bydelivering food for bees that contains the lysing phage to the bee hive.9. The method of claim 7, wherein lysing of the first strain of P.larvae causes a hole in the peptidoglycan of a cell wall of the firststrain of P. larvae and cell membrane of the first strain of P. larvaewhich is externalized after internal pressure force the cell membraneoutside the hole in the cell wall, leading to rupture of the cellmembrane and loss of intercellular components.
 10. The method of claim9, wherein rupture of the cell membrane leads to death of the firststrain of P. larvae.
 11. The method of claim 10, wherein the firststrain of P. larvae that is not P. larvae 2605 deters productivereplication of P. larvae 2605 and the at least two other strains of P.larvae.
 12. The method of claim 9, wherein the first strain of P. larvaethat is not P. larvae 2605 deters productive replication of P. larvae2605 and the at least two other strains of P. larvae.